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Many forms of nucleic acid amplification reactions have been developed in recent years. The first method was the Polymerase Chain Reaction (PCR) which involved repeated cycles of heating to separate the DNA strands, primer annealing to the strands, and primer extension by a DNA polymerase. Product accumulation from the PCR reaction is exponential; that is, the amount of product doubles for every cycle of amplification. Therefore, the expected amount of product may be calculated by the formula (Eff * 2)n where Eff is the efficiency of the primer annealing and primer extension reaction, and n is the number of cycles.
An alternative method for target amplification was developed called NASBA (Nucleic Acid Sequence Based Amplification). This method relies on the concerted action of three enzymatic activities, Reverse transcriptase, RNaseH, and RNA Polymerase, to amplify an RNA target. Reverse transcriptases generally possess an endogenous RNase H activity, which can under the correct conditions, substitute for exogenously added RNase H activity. Primers are first designed which have an RNA polymerase site together with a target recognition sequence. Then, the primers are added to the target nucleic acid together with the three enzyme activities. First, primer binds followed by primer extension across the sequence of interest. The result is a double stranded RNA-DNA hybrid. The RNA portion of the hybrid is digested by the RNase H activity allowing binding of the other primer. The reverse transcriptase activity then extends this primer back across the sequence of interest finishing at the RNA polymerase binding sequence. The RNA polymerase activity then transcribes the sequence of interest making multiple single stranded RNA copies. These RNAs may bind more primers and the cycle continues. Because each transcription step yields 10-100 copies of RNA per copy of template, product accumulates rapidly and logarithmically.
Still, another method has been developed which is called SDA or Strand Displacement Amplification. This method utilizes four primer sequences with two primers binding on either end of the sequence of interest. It also requires a DNA polymerase and a restriction endonuclease (A restriction endonuclease binds to a specific sequence called its recognition site, and then cleaves the DNA a specific sequence). In the first step, nucleic acid strands are heat separated allowing the binding of the first primer pair. The inner primer contains a restriction enzyme site which is non-complementary to the target sequence, while the outer primer binds just upstream of the inner primer. DNA polymerase extends both primers, but extension from the outer primer displaces the newly synthesized inner strand yielding a single strand template for primer binding. Extension reactions are done in the presence of a nucleotide analog (alpha-thio-dATP such that the newly synthesized strands are fully substituted making them immune to cleavage by the restriction endonuclease. However, since the inner primers are not substituted, and the complement of the inner primer is substituted, the restriction enzyme will create a nick within the inner primer sequence by cutting only within the unsubstituted sequence. The nick can act as a priming site for DNA polymerase. In the process of extending the nick, the DNA strands are separated or displaced by the DNA polymerase creating single strand primers which can then bind inner primers for the next round of amplification. Accumulation of product for SDA is therefore exponential since every priming event doubles the amount of product.
Other amplification schemes have been devised, but they all require generating a single strand intermediate that allows primer binding for continued rounds of amplification. While the methods described above have been shown to work well, they do have some drawbacks. PCR requires the use of a thermocycler to obtain rounds of strand separation and primer extension. Furthermore, the process of heating and cooling can be slow resulting in a PCR reaction requiring a few hours to complete from start to finish. NASBA circumvents this issue by being run isothermally, that is at a single temperature. The products are single strand RNA which can be relatively unstable especially if an RNase activity, which are ubiquitous, is inadvertently introduced. RNA products are also generally chemically less stable. Furthermore, the length of the expected product dictates the efficiency of the amplification reaction. This is in part due to the reverse transcriptase activity which tend to be less processive than many DNA polymerases. NASBA reactions also require the addition of high concentrations of both ribonucleotides and deoxyribonucleotides increasing the cost of running a reaction. NASBA reactions are also run at lower temperatures leading to the production of spurious amplification products. In SDA, while the amplification products are DNA, the products are modified by the presence of the alpha-thio-dATP used to inhibit strand cleavage by the restriction endonuclease which may make further manipulation of the product difficult, especially in research applications.
There is a need for improved methods of nucleic amplification. This invention meets those needs.
This invention discloses a method for amplifying any nucleic acid sequence comprising of the steps (i) formation of an intermediate duplex [1] structure from any nucleic acid consisting of a complete double stranded RNA polymerase binding site, a region of sequence to be amplified, and a single stranded RNA polymerase binding site; (ii) binding of a mutant RNA polymerase which utilizes only dNTPS to the RNA polymerase binding site; (iii) transcribing the intermediate duplex to form the first primeness (+) single stranded amplification product [2]; (iv) binding of primer 2 [3] to the primerless (+) single stranded amplification product; (iv) extension of the primer sequence to yield amplification duplex 1 [4]; (v) transcription of amplification duplex 1 to produce the Primerless (xe2x88x92) Strand Single stranded amplification product [5]; (vi) bind primer 1 [6] to the Primerless (xe2x88x92) Strand Single stranded amplification product; (vii) extend Primer 1 by either the mutant RNA polymerase alone or with a second DNA polymerase activity to form amplification duplex 2 [7]; and (viii) transcription of amplification duplex 2 to produce the primeness (+) strand single stranded amplification product [2]. The cycle is continued until one or more of the necessary reaction components are exhausted.
More specifically, this invention is an isothermal amplification method of copying a nucleic acid sequence comprising the steps of:
a. providing an aqueous solution comprising
i. a target nucleic acid for amplification said target comprising a double stranded DNA having a first 5xe2x80x2 end which bears a phage-encoded RNA polymerase recognition site and a second 5xe2x80x2 end which bears a phage-encoded RNA polymerase recognition sequence,
ii. a first and second amplification primer each having a phage-encoded RNA polymerase recognition sequence wherein the first primer is complementary to the 5xe2x80x2 end of the target sequence and the second primer is complementary to the antisense sequence of the 3xe2x80x2 end of the target sequence,
iii. phage-encoded RNA polymerase mutated to recognize and polymerize dNTP and,
iv. an excess of dNTP;
b. repetitively allowing the polymerase to bind to its recognition site and to transcribe a first, short (xe2x88x92) copy strand of the target nucleic acid to yield a multiple copies of a primerless single (+) strand amplification product;
c. creating a first amplification duplex by allowing the second primer to bind to the primeness single (+) strand amplification products of step b and permitting the polymerase to (i) extend the primer to yield a polymerase primed (xe2x88x92) amplification product and (ii) extend the primeness (+) strand to include a polymerase primer complement sequence creating a polymerase recognition site;
d. repetitively allowing the polymerase to bind to its recognition site on the first amplification duplex and to transcribe multiple copies of a primeness single stranded (xe2x88x92) amplification product;
e. creating a second amplification duplex by allowing primer 1 to bind to the primeness single stranded (xe2x88x92) amplification products of step h and permitting the polymerase (i) to extend primer 1 to yield a polymerase primed (+) amplification product and (ii) to extend the primeness (xe2x88x92) strand to include a polymerase primer complement sequence creating a polymerase recognition site; and,
f. repetitively allowing the polymerase to bind to its recognition site on the second amplification duplex and to transcribe multiple copies of a primeness single stranded (+) amplification product.
The above-described method is optionally performed with a T7 RNA polymerase mutant such as Y639F and S641A. The target nucleic acid is optionally derived from a template nucleic acid having a subsequence as the target nucleic acid wherein the method further comprises the steps of: (i) placing the template nucleic acid in an aqueous solution comprising the first and second primers, the mutant phage polymerase and an excess of dNTP and (ii) permitting the polymerase and reactants to yield the target nucleic acid (intermediate duplex) comprising a double stranded DNA having a first 5xe2x80x2 end which bears a phage-encoded RNA polymerase recognition site and a second 5xe2x80x2 end which bears a phage-encoded RNA polymerase recognition sequence. The method can be performed using single stranded DNA as the target nucleic acid. Alternatively with the use of reverse transcriptase the target can be RNA.
In a related method this invention is a logarithmic, isothermal method of copying a nucleic acid sequence from a long strand of nucleic acid comprising the steps of:
a. providing an aqueous solution comprising:
i. template nucleic acid having a target sequence for amplification,
ii. a first and second amplification primer each having a phage-encoded RNA polymerase recognition sequence wherein the first primer is complementary to the 5xe2x80x2 end of the target sequence and the second primer is complementary to the antisense sequence of the 3xe2x80x2 end of the target sequence,
iii. phage-encoded RNA polymerase mutated to recognize and polymerize dNTP and,
iv. an excess of dNTP;
b. allowing the first primers to bind to the template nucleic acid at the 3xe2x80x2 end of the target sequence;
c. creating target: long strand duplex by allowing the polymerase to extend the 3xe2x80x2 end of the primer to create a first, long (+) strand complementary to the target subsequence;
d. displacing the template and the first, long (+) strand;
e. creating an intermediate duplex by allowing the second primer to bind to the long (+) strand at the 3xe2x80x2 end of the target sequence and using polymerase to extend the second primer in a 3xe2x80x2 direction to yield a first, short (xe2x88x92) copy strand bound to the long (+) strand;
f. repetitively allowing the polymerase to bind to its recognition site and to transcribe the first, short (xe2x88x92) copy strand of the intermediate duplex to yield a multiple copies of a primeness single (+) strand amplification product;
g. creating a first amplification duplex by allowing primer 2 to bind to the primeness single (+) strand amplification products of step f and permitting the polymerase to (i) extend the primer to yield a polymerase primed (xe2x88x92) amplification product and (ii) extend the primerless (+) strand to include a polymerase primer complement sequence creating a polymerase recognition site;
h. repetitively allowing the polymerase to bind to its recognition site on the first amplification duplex and to transcribe multiple copies of a primeness single stranded (xe2x88x92) amplification product;
i. creating a second amplification duplex by allowing primer 1 to bind to the primeness single stranded (xe2x88x92) amplification products of step h and permitting the polymerase (i) to extend primer 1 to yield a polymerase primed (+) amplification product and (ii) to extend the primeness (xe2x88x92) strand to include a polymerase primer complement sequence creating a polymerase recognition site; and,
j. repetitively allowing the polymerase to bind to its recognition site on the second amplification duplex and to transcribe multiple copies of a primeness single stranded (+) amplification product.
The methods described herein can comprise a reaction mixture further containing a bumper oligonucleotide which is: (i) able to hybridize to a DNA sequence about or adjacent to the 5xe2x80x2 end of the first long strand and (ii) able to serve as polymerase primer which displaces the first long strand when extended towards the 3xe2x80x2 end of the target nucleic acid.
This invention further comprises a novel composition comprising a double stranded DNA having a first and second end comprising a phage RNA polymerase recognition sequences on both the first and second ends wherein at least one end has a complementary sequence that forms a phage polymerase recognition site [1]. This is termed an intermediate duplex. The composition may also be a double stranded DNA having wherein both ends have phage polymerase recognition sites and the sites may be the same or different. The composition may optionally comprise a signature sequence for a specific genus or species of organism.
This invention also provides for a novel aqueous reaction mixture comprising: i. a target nucleic acid for amplification; ii. a first and second amplification primer [3,6] each having a phage-encoded RNA polymerase recognition sequence wherein the first primer is complementary to the 5xe2x80x2 end of the target sequence and the second primer is complementary to the antisense sequence of the 3xe2x80x2 end of the target sequence; iii. phage-encoded RNA polymerase mutated to recognize and polymerize dNTP; and, iv. an excess of dNTP. The reaction mixture may also comprise target nucleic acid which is a double stranded DNA having a first 5xe2x80x2 end which bears a phage-encoded RNA polymerase recognition site and a second 5xe2x80x2 end which bears a phage-encoded RNA polymerase recognition sequence.
This invention further provides for a kit for amplifying a target nucleic acid comprising a container containing a first primer having a sequence complementary to a 5xe2x80x2 end of the target nucleic acid and a phage polymerase recognition sequence and a container containing a second primer having a sequence which is the anti-complement to the 3xe2x80x2 end of the target nucleic acid and a phage polymerase recognition sequence. The kit may also have a mutant phage polymerase competent to incorporate dNTP into a template nucleic acid. The kit may also have a bumper oligonucleotide which is able to hybridize to a template DNA sequence where that sequence is about or immediately adjacent to the 3xe2x80x2 base of the sequence to which one of the amplification primer binds.
The invention discloses methods to produce the intermediate duplex from any nucleic acid as well as a method based on the use of synthetic DNAs.